Synthesize of Bi2O3/Gln-TSC nanoparticles and evaluation of their toxicity on prostate cancer cells and expression of CASP8, BAX, and Bcl-2 genes

Due to the high prevalence and considerable increase of prostate cancer, finding novel therapeutic compounds for the treatment of prostatic cancer has been the goal of many researches. In this study, we aimed to fabricate the Bismuth oxide (Bi2O3) NPs, functionalized with glutamine (Gln) and conjugated with Thiosemicarbazide (TSC). Then, the anticancer mechanism of the synthesized NPs was investigated using the cellular and molecular tests including MTT assay, Flow cytometry, Caspase-3 activity assay, Hoechst staining and Real Time PCR. The FT-IR and XRD assays confirmed the identity of the synthesized Bi2O3/Gln-TSC NPs. The size range of the synthesized spherical particles was 10–60 nm and the zeta potential was − 23.8 mV. The purity of the NPs was confirmed by EDX-mapping analysis. The Bi2O3/Gln-TSC was considerably more toxic for prostate cancer cells than normal human cells and the IC50 was calculated 35.4 and 305 µg/mL, respectively. The exposure to the NPs significantly increased the frequency of apoptotic cells from 4.7 to 75.3%. Moreover, the expression of the CASP8, BAX, and Bcl-2 genes after exposure to the NPs increased by 2.8, 2.3, and 1.39 folds. Treating the cancer cells with Bi2O3/Gln-TSC increased the activity of the Caspase-3 protein and apoptotic morphological features were observed by Hoechst staining in the treated cells. This work showed that Bi2O3/Gln-TSC has considerable cytotoxicity for prostate cancer cells and could inducing both intrinsic and extrinsic pathways of apoptosis.

www.nature.com/scientificreports/ treatment. Therefore, the preparation of novel nanostructures containing several effective molecules could be a promising method for the introduction of novel and safer anticancer agents for cancer treatment. Bismuth NPs have gained considerable attention in the biomedicine and the cosmetic industry 4 . The bactericidal, fungicidal, antiparasitic and anti-biofilm properties of Bismuth NPs coated with lipophilic dimercaptopropanol NPs have been extensively studied in several papers 5,6 . The in vitro cytotoxicity of Bismuth NPs coated with cellulose nanofibers was evaluated by MTT assay on mouse breast cancer cell lines 7 . Recently, Hamood et al. 8 evaluated the anticancer activity of Bi@PVP NPs on the human MCF-7 breast cancer cell line. The cytotoxic effects of the BisBAL NPs were specifically studied on erythrocytes 9 , epithelial cells 10 , fibroblasts and cancer cells (cervical, prostate, colon in humans) 11 . The generation of intracellular reactive oxygen species (ROS) and activation of cellular pro-apoptotic pathways have been the major cytotoxic features of these NPs that could be employed for cancer chemotherapy [4][5][6][7][8][9][10][11][12] . However, the accumulation in the body and also, cytotoxic side effects are considered the major drawback of them to be used in medical applications.
Conjugation of therapeutic molecules is a novel approach that could be used to increase the efficacy of NPs in cancer treatment and reduce their cytotoxic side effects. Glutamine plays a critical role in cancer metabolism. An excessive uptake and consumption of glutamine by a variety of cancer cells have been reported 13,14 . Therefore, conjugation of nanoparticles to glutamine may increase the efficacy of the complex molecule against cancer cells. Also, glutamine could be used for the functionalization of NPs and their conjugation to therapeutic molecules to increase their anticancer activity 15,16 .
Thiosemicarbazides (TSCs) and their derivatives have shown considerable therapeutic potential as antiviral, antibacterial, and anticancer agents 13 . TSCs could interrupt the replication of cellular nucleic acids and inhibit cell proliferation 14 . Therefore, the conjugation of TSCs to NPs may increase the activity of the complex compared with either agent. In this regard, the present work aimed to fabricate bismuth oxide nanoparticle functionalized with glutamine and conjugated with TSC and to characterize its effect on prostate cancer cells and expression of CASP8, BAX, and Bcl-2 genes.

Synthesis of Bi 2 O 3 and Bi 2 O 3 /Gln-TSC NPs.
To prepare Bi 2 O 3 /Gln NP, at first, 300 mg of Bismuth (III) nitrate and 150 mg of Glutamine (Sigma-Aldrich) were dissolved in 150 mL of dissolved water and the pH was adjusted to 11.0 using 10% NaOH solution. Next, the mixture was heated at 80 °C for 2 h and the resulting dark pellet was harvested, washed, and dried at 70 °C for 8 h (Scheme 1).
For the synthesis of Bi 2 O 3 /Gln-TSC, 500 mg of the synthesized Bi 2 O 3 /Gln and 200 mg of TSC were suspended in 150 mL 96% ethanol and sonicated for 45 min at 40 °C. The mixture was maintained at 40 °C for 24 h with continuous shaking. The prepared Bi 2 O 3 /Gln-TSC NPs were harvested by centrifugation and dried at 70 °C for 8 h 17 .
Physicochemical assays were performed to characterize the prepared Bi 2 O 3 /Gln-TSC. FT-IR analysis was performed using a Nicolet IR 100 FT-IR spectrophotometer in a range of 500-4000 cm −1 . Also, the X-ray diffraction (XRD) pattern of the NP was determined to assess the crystalline structure of the particles (Co-Ka X-radiation, k = 1.79 Å). Scanning (Zeiss-Sigma VP, Germany) and transmission electron microscopy (Zeiss-EM10C, Germany) were used to determine the size range and morphology of the NP. The purity of the Bi 2 O 3 /Gln-TSC was assessed using the EDX-mapping analysis. Further, Zeta potential and DLS analyses (Malvern Instruments Ltd, Ver. 6.32) were used to assess the stability and aggregation degree of the NP.  Flow cytometry analysis. The PC3 cells were treated with Bi 2 O 3 /Gln-TSC NPs at IC 50 concentration.
After 24 h incubation at 37 °C, the cells were washed and treated with propidium iodide and Annexin V (Roche, Germany). Then, flow cytometry analysis was performed (Partec flow cytometry device, Germany) to determine the frequency of apoptotic cells in NPs treated and control cells.
Quantitative PCR (qPCR). The effect of Bi 2 O 3 /Gln-TSC on the expression of Bcl-2, BAX, and CASP8 genes in prostate cancer cells was investigated by SYBR green quantitative PCR assay. The cancer cells (about 5000 cells) were treated with the NPs at IC 50 concentration for 24 h, total RNA of the cells was extracted using the Tri-Zol reagent (Sigma-Aldrich), and cDNA was synthesized by SinaClone cDNA synthesis kit (Iran). The GAPDH gene was used as an internal reference gene. The sequence of the primers used in this study was presented in Table 1. Data analysis was performed according to the 2 −ΔΔCT method 20 .
Activity of Caspase-3. The activity of Caspase-3 protein in Bi 2 O 3 /Gln-TSC treated and control cells was measured using the method described by Salehzadeh et al. 21 . After exposure of PC3 cells with IC 50 concentration of NP, the cells were lysed and the cell supernatant was treated with DEVD-pNA (CASP3C, Sigma-Aldrich). Finally, the optical density was measured at 405 nm.
Hoechst staining. To  Statistical analyses. Significant differences between the NPs treated and control cells were assessed using the SPSS 16.0 software. One-way analysis of variance (ANOVA) along with Post hoc analysis according to Tukey's Test method was used to uncover specific differences between group means, when ANOVA test was significant. The assays were performed in triplicates and p-values less than 0.05 were considered statistically significant.

Results
Physicochemical features of NP. FT 23 . Also, the peaks that appeared at 2θ of 32, 35, and 55.2 contribute to the Glutamine molecule of the fabricated NPs 24 . Considering the amorphous nature of TSC, two broad peaks at 2θ of 40 and 72, and also, the overall reduction of the peak intensity of Bi 2 O 3 /Gln-TSC NPs (compared with the Bi 2 O 3 /Gln), the presence of TSC molecules could be concluded 25 (Fig. 2).
The SEM and TEM images of Bi 2 O 3 /Gln-TSC NPs revealed that the synthesized NPs were almost spherical, with low aggregation, and with a size range of 10-60 nm. Figure 3 presents the electron microscopy images of the fabricated NPs. Moreover, the Zeta potential and hydrodynamic size of Bi 2 O 3 /Gln-TSC were measured − 23.8 mV (Zeta SD = 14.7 mV) and 320 nm (PDI = 0.227), respectively that indicate the proper stability and low aggregation of the NPs. The results were displayed in Fig. 4. The EDX-mapping analysis revealed that the NPs contained Bi, C, N, O, and S atoms and were free of impurities ( Fig. 5 and Table 2).

MTT assay. The cytotoxic effect of Bi 2 O 3 /Gln-TSC for cancer cells and normal human cells was investigated
by MTT assay. According to the results, the Bi 2 O 3 /Gln-TSC NPs were more toxic for cancer cells than HEK293 cells with IC 50 of 35.4 and 350 µg/mL, respectively. Furthermore, the IC 50 of Bi 2 O 3 /Gln NPs on cancer cell line was 74.8 µg/mL. Therefore, the cancer cells were considerably more susceptible to the synthesized NPs than normal human cells. Our results showed that at 6.25 µg/mL and higher concentrations, Bi 2 O 3 /Gln-TSC significantly reduced the viability of cancer cells. The results were presented in Fig. 6.
Flow cytometry analysis. Flow cytometry assay was performed to investigate the effect of exposure to Bi 2 O 3 /Gln-TSC NPs on the apoptosis of prostate cancer cells. Our results showed that after exposure to the NPs, the frequency of apoptotic cells considerably increased compared with the control cells. The frequency of the cells with early and late apoptosis in control PC3 cells was measured 2.53 and 2.17%, respectively, while, after treating the cells with Bi 2 O 3 /Gln-TSC NPs the frequency of early and late apoptosis increased to 19.6 and 55.7%, respectively. Also, about 2.54% of the treated cancer cells with NPs were in necrosis condition and 22.2% of them were alive. In treating the cancer cells with Bi 2 O 3 /Gln NPs the frequency of early and late apoptosis was 28.57 and 5.51%, respectively. Furthermore, about 2.8% of the treated cancer cells with Bi 2 O 3 /Gln NPs were in necrosis condition and 63.2% of them were alive. The results were presented in Fig. 7. Caspase-3 activity assay. The activity of caspase-3 protein in prostate cancer cells was significantly increased than in control cells. As presented in Fig. 9, we observed that the exposure to Bi 2 O 3 /Gln-TSC could significantly increase the caspase-3 activity by 3.6 folds.
Hoechst staining. Hoechst staining assay was performed on the Bi 2 O 3 /Gln-TSC treated and control cells to determine the possible nuclear damages caused form NPs exposure. The results revealed the chromatin condensation and fragmentation and also the appearance of apoptotic bodies as the major outcomes of exposure to Bi 2 O 3 /Gln-TSC NPs (Fig. 10).

Discussion
The frequency of prostate cancer incidence and mortality has been increasing in recent years and thus, finding novel anticancer agents to treat this disease is the goal of many research projects 1 . Nanotechnology products have received great attentions to be used in medicine, especially in cancer treatment. Due to the large surface area, nano-scale compounds could efficiently distribute in the host organs, reach their target sites and exert their cytotoxic effects on the target cells. However, the toxic side-effects of such compounds on the host's organs are the major barrier to their development and application of them in cancer chemotherapy. In this regard, the preparation of complex compounds containing different effective molecules could increase the efficiency and reduce undesirable features of nanotechnology products. In the current work, we aimed to prepare bismuth oxide NPs, functionalize them with glutamine, and conjugate it to TSC. Then, the effect of the Bi 2 O 3 /Gln-TSC NPs on the viability and apoptosis pathway of prostate cancer cells was evaluated. Identity of the fabricated Bi 2 O 3 /Gln-TSC NPs were confirmed using the physiochemical assays. The FT-IR and XRD assays confirmed the proper functionalization and synthesis of the NPs. The zeta potential and XRD revealed the stability and low aggregation of the NPs. Further, the nano-scale size and purity of the NPs were confirmed by the electron microscopy and EDX-mapping analyses.
The MTT assay revealed that the fabricated Bi 2 O 3 /Gln-TSC NPs were considerably more cytotoxic for the prostate cancer cells than normal human cells. Also, Bi 2 O 3 /Gln-TSC NPs were more cytotoxic on cancer cell line than Bi 2 O 3 /Gln NPs. Two main hypotheses could be introduced for the higher susceptibility of prostate cancer cells: (1) high proliferation rate and nutrient intake by cancer cells; (2) higher permeability of cancer cells. Due to the high proliferation rate, cancer cells have considerably higher membrane permeability than normal cells to accelerate cell nutrient intake 26 . However, the increased cell permeability could facilitate the penetration of anticancer drugs into the cell. Moreover, glutamine, as an essential amino acid, is considered an essential nutrient for human cells. Due to the higher metabolic rate, cancer cells seem to have a higher demand for such www.nature.com/scientificreports/ nutrients 27 . The amino acid glutamine plays a key role in the metabolism of highly proliferating cells. During malignant transformation, cancer cells modify the consumption and processing of glutamine to sustain cell growth and proliferation. In some cases, these cancer cells become addicted to glutamine 28 . Thus, targeting the metabolism of glutamine has been developed during last years as a potential strategy against cancer. Therefore, the higher susceptibility of prostate cancer cells than normal cells could be associated with the increased uptake of the Bi 2 O 3 /Gln-TSC NPs into the cancer cells. Previous studies reported that exposure to bismuth NPs could generate radical oxygen species. The generated oxidative stress could damage major cell components, including cell membrane, nucleic acids, enzymes, etc. In addition, previous studies reported that TSC and its derivatives could interrupt the proliferation of eukaryote cells via the inhibition of the enzymes associated with nucleic acid synthesis and replication 14,29,30 . Evaluation of the frequency of apoptosis in the NP treated and control cells showed that Bi 2 O 3 /Gln-TSC considerably increased the frequency of cell apoptosis. The increase of cell apoptosis suggests that Bi 2 O 3 /Gln-TSC could arrest the cell cycle and initiate cell apoptosis. To investigate this hypothesis, the expression of the apoptotic genes and activity of Caspase-3 enzyme in NPs treated cells were investigated. Real-time PCR assay showed that the expression of CASP8, Bcl-2, and BAX genes in NPs treated cells were significantly increased, while exposure to the NPs downregulated the gene. Caspase-8 is an effector molecule in the extrinsic apoptotic signaling pathway. The generation of oxidative stress in the extracellular environment of NP s treated cells could result in the activation of Caspase-8 protein, which triggers the activation of apoptosis pathways via death receptors 31,32 . Therefore, the increased expression of CASP8 gene suggests the upregulation of the extrinsic apoptotic pathway that may be associated with the cytotoxic effects of the NPs. Also, the activity of Caspase-3 enzyme as the activator of the caspase-dependent apoptotic pathway was significantly increased upon exposure to Bi 2 O 3 /Gln-TSC NPs 33 . The increased activity of Caspase-3 enzyme reinforces the role of Caspases in the initiation of the apoptotic pathways in the NPs treated cells.
The expression of the BAX gene in NPs treated cells was increased significantly while exposure to Bi 2 O 3 / Gln-TSC slightly increased the expression of Bcl-2. The BAX protein is regarded as a fundamental effector in www.nature.com/scientificreports/ the intrinsic apoptosis signaling pathways 34 . In contrast, the BCL2 protein mainly acts as the inhibitor of BAX molecules. The considerable increase of the BAX/Bcl-2 ratio could suggest the activation of both intrinsic and extrinsic pathways of apoptosis in NPs treated cells. In accordance with the present results, previous studies have demonstrated that the BAX/Bcl-2 ratio > 1 can induce the apoptosis 35,36 . The Hoechst staining of the cells after treatment with Bi 2 O 3 /Gln-TSC NPs indicated the apoptosis features in the exposed cells. In agreement with the flow cytometry and molecular assays, the Hoechst staining confirmed the role of apoptosis in the death of prostate cancer cells in NPs exposed cells.
This work showed that Bi 2 O 3 /Gln-TSC was highly effective against prostate cancer cells. The cytotoxic potential of Bi 2 O 3 /Gln-TSC could be associated with synergism of all components, including Bi 2 O 3 , glutamine, and TSC. As described above, cancer cells have high proliferation rate and nutrient uptake 26 . Since glutamine is an essential nutrient for human cells, it may facilitate internalization of the Bi 2 O 3 /Gln-TSC into the cancer cells 27 . Inside the cells, other components, including Bi 2 O 3 and TSC could exert their cytotoxic effects by generation of oxidative stress and inhibition of cell enzymes, respectively. Therefore, the synergistic effect of Bi2O3/Gln-TSC components could induce apoptotic signaling pathways in prostate cancer cells.

Conclusion
In this study, Bi 2 O 3 /Gln-TSC NPs were fabricated and its anticancer effect on prostate cancer cells was investigated. Our results revealed that the synthesized NPs were considerably more cytotoxic for cancer cells than normal human cells and the activation of cell apoptosis through both extrinsic and intrinsic pathways was suggested as the main cytotoxic mechanism of the NPs. Further investigations in in-vivo models could elucidate the potential of Bi 2 O 3 /Gln-TSC NPs to be applied in anticancer chemotherapy.  www.nature.com/scientificreports/